A protein switch alters its behavior in response to a change in environmental conditions, such as the presence of a specific ligand. Recently, a protein switch was engineered through combinatorial library searching to couple the function of maltodextrin binding protein (MBP) and TEM-1 3-lactamase (BLA). A conformational change in the MBP upon binding to maltose results in a switch that displays a 280-fold increase in enzymatic activity in the presence of maltose. Despite the extensive research done with this fusion system, the structural mechanism remains unknown. Furthermore, there are no computational approaches for designing this type of switch and the underlying design principles are not well understood. The research set forth in this proposal outlines a strategy for structure prediction and design of domain insertion molecular switches. A mechanistic, structural understanding developed by the proposed work will allow for the rational and computational improvement of existing switches as well as design of new switches. The aims of the proposed research are to (1) develop and validate a structure prediction algorithm for determining the structure of domain insertion proteins;(2) generate structural models of MBP-BLA fusion protein and its allosteric mechanism;(3) computationally design and experimentally test novel MBP-BLA fusion protein switches;and (4) use this information to create new switches that couple specific functions of multiple proteins. The ability to create such switches through engineering or modification of existing proteins has a variety of transcending biotechnological applications, such as targeted drug delivery, inhibition of enzyme activity, or development of novel molecular sensors. This research is relevant to public health because it will provide general computational tools to discover the structural mechanism of protein switches that have applications to medicine. This will provide a foundation needed to be able to effectively couple the function of two proteins that can be useful for manipulating cells via regulatory pathways, interfering with the signaling of the immune system, or restoring proper function in malfunctioning diseases (e.g. cancer or arthritis).